The transcription factor NF-κB is an established regulator of numerous genes important in the inflammatory response. More recently, activation of NF-κB has been shown to have a role in many aspects of oncogenesis including control of apoptosis as well as regulation of cell cycling and cell migration (Yamamoto et al., J. Clin. Invest. 2001, 107, 135; Baldwin, A. S. J. Clin. Invest. 2001, 107, 241). Activated NF-κB has been observed in many cancers and is especially important in metastasis (Andela et al., Clin. Orthop. Relat. Res. 2003, 415 (suppl), S75).
NF-κB is a collective name for dimeric transcription factors comprising members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-κB is also commonly referred to as, for example, NFκB and NFκB, with the abbreviations being used interchangeable. Five members of the mammalian Rel family are known: RelA (p65), RelB, c-Rel, NF-κB1 (p50) and NF-κB2 (p52) (Baldwin, A. S., Annu. Rev. Immunol. 14, 649 (1996)). The five members to the NFκB family are distinguished by the presence of a Rel homology domain. Each NFκB member is retained in the cytosol as a complex, the most prevalent of which is a dimer consisting of the two subunits, p65 and p50. Any homo- and heterodimer is considered NF-κB, although the most commonly found in activated cells, RelA/NF-κB (p65/p50) heterodimer, is often referred to as “classic” NF-κB. All Rel proteins contain a Rel homology domain (RHD) that is responsible for dimer formation, nuclear translocation, sequence-specific DNA recognition, and interaction with I-κB proteins. RelA, RelB and c-Rel also contain transactivation domains required for the recruitment of transcriptional machinery, and thus represent transcriptionally active components of NF-κB. FIG. 1A provides a pictorial representation of the NF-κB activation cascade.
NF-κB activation is controlled by an interaction with a family of inhibitors proteins known as I-κB. The I-κB family includes I-κBα, I-κBβ, I-κBγ, I-κBε, p100, p102, and Bcl-3 (Whiteside et al., Semin. Cancer Biol. 8, 75 (1997)). All I-κBs share three common structural features: an N-terminal regulatory domain, which is responsible for a signal-dependent I-κB proteolysis, a core domain composed of six or seven ankyrin (ANK) repeats mediating an interaction with Rel proteins, and a C-terminal domain containing a PEST motif implicated in basal I-κB turnover.
In unstimulated cells, NF-κB resides in the cytoplasm as an inactive NF-κB-I-κB complex. I-κB binding hinders recognition of the NF-κB nuclear localization signal by nuclear import machinery, thus retaining NF-κB in the cytoplasm. Stimulation of cells releases active NF-κB, which is now free to enter the nucleus and activate transcription. Release of NF-κB is generally mediated by the degradation of I-κB.
Phosphorylation of IκB by IκB kinase (IKK) in response to an array of signals leads to degradation of IκB and the release of NFκB. Free NFκB is translocated to the nucleus where it binds to promoter regions of DNA resulting in the activation of a battery of genes, including pro-inflammatory genes (cytokines IL1 and TNFα; chemokines; stress response genes; and pro-inflammatory enzymes including iNOS, COX-2 and MMP-9). Compounds inhibiting the activation of NFκB can be directed at IKK or at NFκB. IKK inhibitors will prevent phosphorylation of IκB whereas direct inhibitors of NFκB may block NFκB-DNA interactions. Karin et al., (2004) Nat Rev Drug Discov 3, 17-26. The inducible degradation of I-κB occurs through consecutive steps of phosphorylation, ubiquination, and proteosomal degradation. I-κB processing is controlled by three large multi-protein complexes: IKK or signalsome, I-κB ubiquitin ligase, and 26S proteosome (Makarov, S. S., Mol Med Today. 6, 441-8 (2000)). Whereas the I-κB ubiquin ligase and the 26S proteosome are constitutively active, IKK activity is induced upon stimulation. Various stimuli, including inflammatory cytokines, mitogens, viral proteins, and stress, can activate IKK, thereby inducing phosphorylation of two critical serine residues of I-κB. The phosphorylation of I-κB targets it for rapid ubiquination and proteosomal degradation.
There are two IKK's, designated IKKα and IKKβ, that exist in a complex called the IKK signalsome. Also included in the complex are the IKK-associated protein (IKAP) and NEMO (also called IKKγ). There are many upstream regulators of the IKK signalsome that have been identified and could be useful “targets” for suppression of IKK expression and, ultimately, NFκB expression. Thus, compounds that prevent the phosphorylation of IκB (and therefore prevent the activation of NFκB) may act directly on one or more members of the IKK signalsome or may inhibit upstream kinases, such as SFK or any other such family of kinases. This complicates structure-based design of potential drugs to prevent activation of NFκB, especially because crystal structures of members of the IKK signal some are not available. It is noteworthy that there are also IKK-independent pathways for activation of NFκB.
Most available evidence suggests that IKKβ is the canonical pathway for NFκB activation and that IKKα functions in special circumstances. Karin et al., (2004) Nat Rev Drug Discov 3, 17-26. Whereas binding to IκBβ effectively sequesters NFκB in the cytoplasm, binding to IκBα does not preclude nuclear translocation. In fact, the NFκB-IκBα trimeric complexes shuttle between the cytoplasm and the nucleus. The source of this difference is that binding of IκBβ to a p50/p65 complex blocks NLS located on both NFκB subunits, whereas binding to IκBα blocks only the p65 NLS. Thus, NFκB-IκBα complexes contain both an exposed functional NLS and several nuclear export signals (NES) found in the N-terminal domain of IκBβ and in the activation domain of p65. The functions of both NLS and NES result in this shuttling between the cytoplasm and the nucleus. However, multiple NES seem to dominate, resulting in a primarily cytoplasmic localization of NFκB-IκBα complexes. When nuclear export is blocked with leptomycin B (LMB), the complex accumulates in the nucleus. Since IκBα is the most prevalent IκB isoform, in most resting cells the majority of NFκB protein is located in the cytoplasm bound to IκBα. Inflammatory stimuli, such as IL-1 treatment, leads to activation of IKK activity, phosphorylation of IκBα on serine 32 and 36, recognition of IκBα by the E3 ubiquitin ligase, IκBα ubiquination, degradation of IκBα by the 26S proteasome, and release of NFκB. The two exposed NLS on NFκB subunits then cause nuclear translocation of the transcription complex. However, numerous studies have now documented states where NFκB activation occurs in the absence of IκBα degradation.
For many stimuli, including interleukin-1 (IL-1), and tumor necrosis factor α (TNF-α), I-κB degradation and NF-κB nuclear translocation are necessary, but not sufficient, for the induction of NF-κB dependent transcription. The ability of NF-κB to initiate transcription depends on interactions with different transcriptional co-activators (Schmitz et al., J. Biol. Chem. 270, 7219-7226 (1995). Although regulated independently, the pathways controlling I-κB degradation and NF-κB transcription function act in synergy with the activation of NF-κB mediated transcription.
NF-κB appears to play a pivotal role in both initiation and perpetuation of chronic inflammation. CD4+ T cells are a trigger of immune inflammation, and NF-κB appears to be an important mediator of antigen-induced T-cell activation. Secreted products of activated T cells and direct cell-cell contacts cause activation of macrophages, fibroblasts, and endothelial cells. Once established, autocrine/paracrine loops of inflammatory cytokines and growth factors are capable of maintaining the activation of non-immune cells within the lesion, thereby perpetuating the chronic inflammatory process. Persistent NF-κB activation has been found in many chronic inflammatory diseases, including rheumatoid arthritis, asthma, inflammatory bowel disease, ulcerative colitis, and atherosclerosis (Barnes et al., New Engl. J. Med. 336, 1066-1071 (1997).
Additionally, the evidence that links activation of NF-κB to oncogenesis is compelling. NF-κB is activated by a number of viral transforming proteins (Hiscott et al., J. Clin. Invest. 2001, 107, 143), and inhibition of NF-κB activation through expression of a dominant negative IKK can block cell transformation (Arsura et al., Mol. Cell Biol. 2000 20, 5381). NF-κB activation protects cells from apoptosis induced by cancer chemo-therapeutics and oncogenes (Barkett et al., Oncogene 1999, 18, 6910), and activation of NF-κB promotes expression of metastatic factors (Baldwin, A. S. J. Clin. Invest. 2001, 107, 241). NFκB activation results in up-regulation of cyclin D1, a cell cycle regulator that is up-regulated in many tumors. NFκB is constitutively expressed in many cancer cell lines. Additionally, a number of dietary chemopreventive compounds such as flavonoids, curcumin, and reserveratol block activation of NFκB. Further, the expression of interleukin 8 (IL-8) which has been identified as a key factor in both angiogenesis and metastasis is very dependent on NFκB activity.
NF-κB is active in many tumors, and expression of NF-κB-responsive genes provide cancer cells with distinct survival advantages that inhibit cancer treatment. NF-κB is constitutively activated in many cancer cells, and NF-κB may also be conditionally activated in both cancer cells and stromal cells by the tumor microenvironment. Normally, NF-κB activation is prevented by binding to inhibitor (IκB) proteins, the most prevalent being inhibitor of NF-κB alpha (I-κBα). In response to inflammatory cytokines, the release of NF-κB is triggered by phosphorylation of I-κBα on serines 32 and 36, resulting in ubiquination and degradation of I-κBα protein. However, in cancer cells subjected to environmental conditions such as hypoxia, nutrient starvation, or X-rays, NF-κB activation is caused by phosphorylation of I-κBα on a tyrosine residue (Tyr42) by Src family kinases (SFKs). Thus, NF-κB activation via IκBα Tyr42 phosphorylation is expected to occur in solid tumors due to constitutive activation of SFKs such as the Src oncogene in response to the hypoxic and nutrient poor nature of the tumor microenvironment, or due to radiation treatment of the tumor.
NFκB was first identified as the nuclear factor in mature B-lymphocytes that binds to an 11 bp element (GGGACTTTCC) within the κ-light chain gene enhancer, but it was soon realized that NFκB is not a B-cell-specific transcription factor. A wide variety of environmental stimuli and stresses lead to the formation of active NFκB complexes within almost every cell type, and NFκB activation mediates the transcription of over 180 target genes.
There are several NFκB crystal structures for use in structure-based drug design including a human NFκB-DNA structure. However, compounds that have been reported to inhibit activation of NFκB have generally been suggested to work at the level of IKK, rather than to interfere with NFκB-DNA interactions or with NFκB dimerization to prevent its interaction with DNA. Given the mechanisms of suppression and expression of NFκB, compounds inhibiting the activation of NFκB can be directed at IKK, SFK, or other kinases at NFκB-DNA interactions. Kinase inhibitors will prevent phosphorylation of IκB where direct inhibitors of NFκB may block NFκB-DNA interactions. For example, it has been shown recently that a new class of retinoid-related drug candidates inhibits IKK directly. Bavon et al., (2003) Mol Cell Biol 23, 1061-1074. By comparison, a synthetic derivative of the fungal metabolite jesterone, which blocks activation of NFκB, was shown to inhibit a kinase involved in phosphorylation and activation of IKK (β-subunit). Liang et al., (2003) Mol Pharmacol 64, 123-131. It appears, therefore, that inhibition of one or more of the kinases associated with the IKK signalsome may be a promising route to the development of new therapeutic agents that work through blocking the activation of NFκB. Further, because NFκB responsive genes can promote angiogenesis, cell motility and invasion, and block apoptotic cell death, this mechanism represents a considerable obstacle to cancer treatment. Therefore, there is a greatly felt need for development of small molecule inhibitors of NFκB expression. Particularly, but not exclusively, inhibitors of IκBα Tyr42 phosphorylation have vast potential to serve as adjuvant cancer therapeutics.
Activator Protein-1 (AP-1) is another protein transcription factor found in mammalian cells. AP-1 like NF-κB is a prosurvival and pro-inflammatory protein. AP-1 is an established regulator of numerous genes important in a variety of cellular processes including cell growth regulation, differentiation and proliferation (Angel et al., Cell 1987, 49, 729-739). Growth factors, hormones, tumor promoters and oncogenes regulate AP-1 binding to DNA (Bernstein et al., Science 1989, 244, 566-569). Activated AP-1 has been shown to play a role in apoptosis, angiogenesis and metastasis (Kang et al., Am. J. Pathol. 2005, 166(6), 1691-1699) and is also involved in many diseases including cancer, diabetes and Alzheimer's disease. AP-1 is also associated with the production of metalloproteinases. Collagenases, a class of metalloproteinases, are known to contain AP-1 response elements in their DNA promoters (Kang et al., Am. J. Pathol. 2005, 166(6), 1691-1699). The combination of these factors makes AP-1 crucial to many oncogenic processes.
AP-1 consists of 18 dimeric combinations of the families Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) (Young et al., Trends Mol. Med. 9(1), 36-41 (2003)). Of the dimeric possibilities are Jun-Jun homodimers and Jun-Fos heterodimers. Jun dimers bind tightly to AP-1 DNA recognition elements (Angel et al., Cell 49, 729-739 (1987)). Fos-Fos homodimers are unstable and not readily formed but can bind to DNA by forming heterodimers with Jun proteins (Ziegler et al., J. Nutr. 2004, 134, 5-10). The most common dimer is a heterodimer consisting of c-Jun and c-Fos. Also associated with the Jun and Fos families are Jun dimerization partners and activating transcription factors (ATF's) (Angel et al., Biochim. Biophys. Acta 1072, 129-157 (1991)).
In normal tissues, the AP-1 component c-Fos is found only in small concentrations but cytosolic levels are rapidly increased when the cell is induced by mitogenic stimuli (Muller et al., Nature 1983, 304, 454-456). c-Jun, another AP-1 component, plays an important role in the regulation of cellular proliferation (Karin et al., Curr. Opin. Cell Biol. 1997, 9(2), 240-246). When c-Jun and c-Fos become unregulated in the body, abnormal cell proliferation occurs leading to cellular transformations. c-Jun is known to be essential in tumor promotion in several cell lines (Jochum et al., Oncogene 2001, 20(19), 2401-2412; Orlowski et al., Trends Mol. Med. 2002, 8,385-389; Pain, Eur. J. Biochem. 1996, 236, 747-771; Karin et al., Nat. Rev. Cancer 2002, 2(4), 301-310; Dhar et al., Mol. Cell. Biochem. 2002, 234-235, 185-193). c-Fos is also involved in the conversion of cells from benign to malignant (Dong et al., Proc. Natl. Acad. Sci. USA 1994, 91, 609-613; Greenhalgh et al., Cell Growth Differ. 1995, 6, 579-586) and is essential in tumor progression (Saez et al., Cell 1995, 82(5), 721-732). In general, the activation of both NF-κB and AP-1 are required for tumor promotion and progression.
The AP-1 activation cascade can be induced by TNFα, okadaic acid, 12-O-tetradecanoylphorbol-13-acetate (TPA), UV light (Young et al., Trends Mol. Med. 9(1), 36-41 (2003)), cytokines, mitogens, phorbol esters, growth factors, environmental and occupational particles, toxic metals, intracellular stresses, bacterial toxins, viral products and ionizing radiation (Fontecave et al., FEBS Lett. 421, 277-279 (1998)). In general, AP-1 is activated primarily through mitogen-activated protein kinase (MAPK) cascades (Kundu et al., Mutat. Res. 2004, 555, 65-80). MAPK's are composed of MAPK itself and MAPK kinase, also called MAPK-extracellular signal regulated kinase (MEK) (Wilkinson et al., Genes Dev. 1998, 12, 1391-1397). MAPK's are activated by cytokines, hormones and stress-inducing agents (Blenis, Proc. Natl. Acad. Sci. USA 1993, 90(13), 5889-5892). In general, the same factors that stimulate NF-κB also stimulate AP-1.
MAPK or MEK can phosphorylate additional kinases including extracellular regulating kinases (ERK's), c-Jun N-terminal kinase (JNK) and p38 MAPK (Baker et al., Mol. Cell. Biol. 1992, 12(10), 4694-4705; Davis, J. Biol. Chem. 1993, 268(20), 14553-14556). JNK activates the c-Jun protein and ERK activates a protein called Elk-1 both by phosphorylation. c-Jun then binds to DNA along with an ATF to activate genes that produce more of the Jun family in a positive feedback loop (Thevenin et al., J. Biol. Chem. 1991, 266(15), 9363-9366). Elk-1 also binds to DNA with a serum response factor (SRF) to activate genes that produce the Fos family. The Jun and Fos protein families are then activated by JNK and c-Fos-regulating kinase (FRK) respectively. The activated families can now dimerize, bind to DNA and activate gene expression that adversely affects cellular processes. A pictorial representation of AP-1 activation is shown in FIG. 1B.
AP-1 proteins and their activating kinases are related to NF-κB. AP-1 proteins are known to interact with the p65 subunit of NF-κB (Li et al., Mol. Carcinog. 29(3), 159-169 (2000)). MAPK's are known to phosphorylate I-κB (Adler et al., EMBO J. 18, 1321-1334 (1999)). Because AP-1 and NF-κB responsive genes can promote angiogenesis, cell motility and invasion, and block apoptotic cell death, activation of these genes and their products may result in cancerous or precancerous growth. Moreover, AP-1 and NF-κB responsive genes can promote inflammation, activation of these genes and their products may result in greater inflammation in diabetics and others. Therefore, there is a greatly felt need for development of small molecule inhibitors of AP-1 or NF-κB activation.
Alzheimer's Disease
Alzheimer's disease (AD), the most common cause of dementia in elderly populations, currently afflicts almost 5 million people in the U.S., and this number is estimated to increase to 15 million by 2050. Hebert et al., (2003) Arch Neurol 60, 1119-1122. Most AD is sporadic with multiple risk factors, while some 10-15% is familial. It is well accepted that excessive production or diminished clearance of the Aβ peptide derived from the amyloid precursor protein (APP) is an essential factor in the etiology of AD. This is supported by studies of genetic mutations in APP in experimental animal models of AD as well as from studies of the genetics of familial AD. Selkoe et al., (2000) Annu Rev Genomics Hum Genet 3, 67-99.
There are two major neuropathological signatures of AD: extraneuronal amyloid plaques and neurofibrillary tangles. The plaques primarily consist of Aβ aggregates while the tangles consist of hyperphosphorylated tau protein. The exact mechanism by which these aggregates cause neuronal cell death remains to be established. However, considerable recent evidence points towards a major role for oligomeric forms of Aβ which are neurotoxic and can diffuse. Soluble Aβ is found in CSF of AD patients and correlates better with severity of disease than does the quantity of plaques. Kim et al., (2003) FASEB J 17, 118-120; McLean et al., (1999) Ann Neurol 46, 860-866.
There are other common features of AD including the presence of chronic inflammation. The inflammatory response in brain is directed by activated microglia and reactive astrocytes. In normal brain, microglia are not activated. Under these conditions, neither pro-inflammatory signals nor reactive oxygen/nitrogen species (ROS/RNS) are formed. McGeer et al., (2003) Prog Neuropsychopharmacol Biol Psychiatry 2, 741-749. However, when microglia become activated in response to various insults, there is up-regulation of a number of surface receptors that promote phagocytotic activity by microglia. In addition, pro-inflammatory signals are released including interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) as well as ROS/RNS, thus contributing to the oxidative stress associated with AD. Activated microglia also associate with amyloid plaques. Microglia isolated from AD brain can scavenge Aβ. Rogers et al., (2002) Glia 40, 260-269. The considerable literature on the role of microglia in AD suggests that activation of microglia may contribute initially to clearance of Aβ aggregates, but that the chronic activation of microglia observed in AD leads to the neuropathological changes in the AD brain. Griffin et al., (1998) Brain Pathol 8, 65-72. Activated microglia also contribute to hyperphosphorylation of tau with development of neurofibrillary tangles, as well as to recruitment of activated astrocytes into the Aβ plaques. Kitazawa et al., (2004) Ann NY Acad Sci 1035, 85-103.
It is now recognized that Aβ can increase the inflammatory response by activation of microglia and that the inflammatory response can contribute to Aβ deposition. Consequently there has been interest in hindering microglial activation as an approach to breaking this pathological cycle. Aisen (1997) Gerontology 43, 143-149. Since activation of microglial results in release of ROS/RNS, attention has focused on use of anti-oxidants such as vitamin E. There are conflicting reports of the effects of anti-oxidants on development of AD, some supporting a role for anti-oxidants (Engelhart et al., (2002) JAMA 287, 3223-3229) and others not supporting a role (Laurin et al, (2004) Am J Epidemiol 159, 959-967). Activation of microglia increases the oxidative burden in affected brain regions. However, how significant this increase is in contributing to neurodegeneration is not known. The field of anti-oxidant treatment of AD will need further controlled trials to assess this question.
Another area that has produced conflicting reports is the use of anti-inflammatory drugs, especially non-steroidal anti-inflammatory drugs (NSAIDS), in treatment of AD. COX-2, the inducible form of cyclooxygenase found in neurons and other cells and the source of pro-inflammatory eicosenoids, is up-regulated in AD brains. Yasojima (1999) Brain Res 830, 226-236. Overexpression of human COX-2 in mice results in age-related cognitive decline as well as neuronal apoptosis and astrocyte activation. Andreasson et al., (2001) J Neurosci 21, 8198-8209. The epidemiology studies of use of COX inhibitors (i.e. NSAIDs) by AD patients suggest that NSAID therapy may be useful. McGeer et al., (2003) Prog Neuropsychopharmacol Biol Psychiatry 2, 741-749. However, controlled clinical trials have been disappointing. These conflicting results may reflect the fact that the epidemiology studies begin with normal subjects and then assess risk of developing disease and whether this risk correlates inversely with drug use, whereas the clinical trials begin with subjects who have AD and look for improvement upon treatment. Other studies suggest that only a limited group of NSAIDs are effective and that these NSAIDs influence multiple targets in addition to COX-2. Gasparini et al., (2005) Brain Res Rev 48, 400-408. Animal model studies suggest that the dosing level of NSAID that is clinically feasible may not be sufficient to produce a pharmacological dose at the sites of plaque formation in AD brains. Cole et al., (2004) Ann NY Acad Sci 1035, 68-84.
Another area of interest in AD drug development focuses on signaling pathways that regulate expression of pro-inflammatory genes. Aβ stimulation of microglia results in up-regulation of the expression of TNFα and IL1 that is at least partly NFκB-dependent. Combs et al., (2001) J. Neurosci. 21, 1179-1188. IL1 is known to affect the expression of over 90 genes including those for cytokines, cytokine receptors, tissue remodeling enzymes and adhesion molecules. O'Neill (1995) Biochim Biophys Acta 1266, 31-44. The mechanism for IL1 action involves activation of an IL1 receptor-mediated signal transduction pathway which leads to activation of NFκB. O'Neill et al., (1998) J Leukoc Biol 63, 650-657. Thus NFκB is involved both in up-regulation of IL1 and in expression of the multiple genes regulated by IL1. These observations make inhibition of NFκB an attractive target for control of IL1-responsive genes in brain inflammation.
Diabetes
In 1998, it was suggested that the innate immune system is activated in diabetes, leading to a chronic inflammatory state that contributes to the disease process (Pickup et al., 1998, Diabetologia 41:1241-1248). More recently, there has been considerable support not only for an inflammatory contribution to diabetes but also to diabetic complications (Navarro et al., 2005, Nephrol Dial Transplant 20:2601-2604; Pillarisetti et al., 2004, Expert Opin Ther Targets 8:401-408). Specifically, pro-inflammatory cytokines play a major role in microvascular complications. Endogenous production of TNF-α in vascular tissue is accelerated in diabetes where it contributes to increased vascular permeability in diabetic neuropathy (Satoh et al., 2003, Exp Diabesity Res 4:65-71). Both TNF-α and IL-1 expression are increased in diabetic retina where chronic low-grade inflammation appears to contribute to retinopathy (Joussen et al., 2002, FASEB J 16:438-440). Likewise, diabetic nephropathy is associated with expression of inflammation markers such as CRP, fibrinogen and IL-6, and with increased expression of adhesion molecules such as ICAM-1, which promote inflammation by increasing leukocyte adherence and infiltration (Dalla Vestra et al., 2005, J Am Soc Nephrol 16:S78-S82). The responses to these pro-inflammatory cytokines are especially prominent in endothelial cells (EC). Moreover, the response of EC to these cytokines commonly involves signaling through transcription factor NF-κB (Mohamed et al., 1999, BioFactors 10:157-167).
Oxidative stress has consistently been shown in experimental models of diabetes (Mohamed et al., 1999, BioFactors 10:157-167). Multiple mechanisms are involved that produce oxidative stress in EC in response to hyperglycemia, including: 1) protein glycosylation leading to AGE that trigger ROS production upon binding to the AGE receptor (RAGE) (Wautier et al., 2004, Circ Res 95:233-238); 2) glucose auto-oxidation (Ceriello, 1997, Diabet Med 14:S45-S49); 3) accelerated metabolism of glucose through the aldose reductase/polyol pathway which consumes NADPH (Srivastava et al., 2005, Endocrin Rev 26:380-392); 4) uncoupling of oxidative phosphorylation and of endothelial NO synthase (eNOS) (Satoh et al., 2005, Am J Physiol Renal Physiol 288:F1144-F1152); 5) activation of specific isoforms of PKC (Ahmed et al., 2005, Curr Drug Targets 6:487-494); 6) increased flux through the hexosamine pathway (Schleicher et al., 2000, Kidney Int Suppl 77:S13-S18); and 7) exposure to angiotensin II (Yamagishi et al., 2005, FEBS Letters 579:4265-4270). Activation of NF-κB is often observed in response to these stresses. For example, exposure of EC to AGE generates ROS through activation of NADPH oxidase which then activates NF-κB followed by up-regulation of NF-κB-dependent cytokines and adhesion molecules (Wautier et al., 2001, Am J Physiol Endocrinol Metab 280:E685-E694). Angiotensin II can augment this process through crosstalk with the AGE-RAGE system, again involving NF-κB (Yamagishi et al., 2005, FEBS Letters 579:4265-4270). High glucose can induce EC apoptosis through a PI-3-kinase-regulated expression of COX-2; this was shown to involve ROS and the NF-κB-regulated expression of COX-2 (Sheu et al., 2005, Arterioscler Thromb Vasc Biol 25:539-545). There has been considerable interest in a role for poly(ADP)-ribose polymerase (PARP) in EC dysfunction. PARP directly interacts with both the p50 and p65 subunits of NF-κB, suggesting that the role of PARP activation in diabetic complications is, at least in part, due to its interaction with NF-κB (Zheng et al., 2004, Diabetes 53:2960-2967). Glucose-induced activation of NF-κB in EC is prevented by inhibitors of PKC, suggesting that the role of PKC in triggering the expression of pro-inflammatory cytokines is through downstream activation of NF-κB (Pieper et al., 1997, J Cardiovasc Pharmacol 30:528-532). There has also been considerable interest in mitochondria-derived ROS (specifically superoxide) produced in response to hyperglycemia and the relationship between these ROS and enhanced flux through the polyol pathway and the hexosamine pathway, PKC activation, and intracellular generation of AGE, all of which can be prevented by inhibiting the formation of mitochondria-derived ROS (Nishikawa et al., 2000, Nature 404:787-790). The activation of these biochemical pathways appears to be due to ROS-induced activation of PARP, which results in inactivation of glyceraldehyde-3-phosphate dehydrogenase and subsequent accumulation of glycolytic intermediates that promote these pathways (Araujo et al., 2001, Mem Inst Oswaldo Cruz 96:723-728). It is noteworthy that inhibiting the production of mitochondria-derived ROS also prevents the activation of NF-κB (Du et al., 2003, J Clin Invest 112:1049-1057), which may be related to the activation status of PARP. Clearly, activation of NF-κB appears to be a general feature of EC that are stressed by factors related to diabetic complications, suggesting a central role for NF-κB in EC dysfunction, especially as the key regulator of pro-inflammatory cytokines, adhesion molecules and extracellular matrix components, all of which are major players in diabetic microvascular complications.
The signaling mechanisms involved in inflammation that contributes to diabetes are under investigation, and are described by Wellen et al. (Wellen et al., J. Clin. Invest., 115, 1111-1119). This research indicates that inflammatory signaling pathways can be activated by metabolic stress or extracellular signaling molecules, and that endoplasmic reticulum stress (ER stress) leads to the activation of inflammatory signaling pathways and thus contributes to insulin resistance. Ozcan et al., Science, 306, 457-461 (2004). For example, several serine/threonine kinases are activated by inflammatory or stressful stimuli that contribute to inhibition of insulin signaling, including c-Jun N-terminal kinase (JNK) and I-κB kinase (IKK). The three members of the JNK group of kinases (JNK-1, -2, and -3) belong to the MAPK family and regulate multiple activities, in part through their ability to control transcription by phosphorylating activator protein-1 (AP-1). Loss of JNK1 has been shown to prevent the development of insulin resistance and diabetes in both genetic and dietary models of obesity. Hirosumi et al., Nature, 420, 333-336 (2002).
A model of the overlapping metabolic and inflammatory signaling and sensing pathways in adipocytes and macrophages that influence diabetes and inflammation is provided by FIG. 2. As shown in FIG. 2, signals from various mediators converge on the inflammatory signaling pathways, including the kinases JNK and IKK. These pathways lead to the production of additional inflammatory mediators such as NF-κB and AP-1 through transcriptional regulation as well as to the direct inhibition of insulin signaling. Opposing the inflammatory pathways are transcriptional factors from the PPAR and LXR families, which promote nutrient transport and metabolism and antagonize inflammatory activity.
Glutathione S-Transferase
Glutathione S-transferases (GSTs) are a superfamily of enzymes classified into eight gene families. Many GSTs are also classified as phase II detoxification enzymes that catalyze the conjugation of glutathione to a wide variety of electrophiles as the first step in elimination of xenobiotics. However, GSTs also exhibit numerous family-specific functions, some but not all of which involve glutathione. For example, GSTP1-1, which is the main member of the “pi” family and is the most widely distributed GST, is important as both a detoxification enzyme and in signal regulation through its protein-protein interactions with c-Jun N-terminal kinase (JNK), a kinase that is important in the stress response and apoptosis. Thus, up-regulation of GSTP1-1 serves to protect cells from apoptosis-inducing stress by inhibiting JNK. Notably, the promoter for GSTP1-1 contains NFκB-binding sites. It is important to understand that oxidative stress leads to modification of critical cysteine residues in GSTP1-1, resulting in the release of JNK and initiation of apoptosis. Therefore, it is known that tumors that over-express GSTP1-1 are resistant to stress-induced apoptosis, and the presence of GSTP1-1 assists in the prevention of apoptosis.
Glutathione S-Transferase P1-1 (GSTP1-1) thus has two distinct functions which contribute to the survival of cancer cells. First, GSTP1-1 detoxifies xenobiotic electrophiles, including some cancer drugs, by catalyzing the conjugation of glutathione, thereby contributing to drug resistance. It is involved in eliminating toxic molecules from the cell including drugs that are supposed to be assisting the cell in fighting diseases, and has been implicated in the development of drug resistance in a variety of cancers. Elevated levels of GSTP1-1 are found in numerous cancer cell lines and tumors, including, among others, breast cancers, prostate cancers, and leukemias that are resistant to a range of anti-cancer drugs. It is known in the art that GSTP1-1 positive breast tumors are more aggressive than GSTP1-1 negative tumors and have a poorer prognosis. For example, the MCF7 breast cancer cell line, which is a GSTP1-1 expressing line, was shown to develop resistance to a number of drugs when the cells were transfected with GSTP1-1. It is also known in the art that ovarian cancer cell lines that over-express GSTP1-1 are resistant to taxol and doxorubicin. In fact, GSTP1-1 has also been used as a prognostic tool in invasive breast cancer. Over-expression of GSTP1-1 has been shown to be a marker of poor outcome in breast cancer and advanced non-Hodgkin's lymphoma. And second, because GSTP1-1 also inhibits the pro-apoptotic factor c-Jun N-terminal kinase (JNK), it promotes the pro-survival state.
A number of studies support the idea that inhibitors of GSTP1-1 may have therapeutic potential in the treatment of cancer. If GSTP1-1 can be inhibited, then known cancer therapeutics would not be eliminated from the cell. In one study, inhibition of GSTP1-1 by the glutathione conjugate of doxorubicin induces apoptosis in rat hepatoma cells. Also, ethacrynic acid, a broad-spectrum inhibitor of glutathione S-transferases, provides a therapeutic advantage when combined with other agents. Ethacrynic acid, however, is a potent diuretic; this along with its lack of GST isozyme selectivity precludes the development of ethacrynic acid as an anti-cancer therapeutic. A number of peptidomimetic inhibitors that are selective for GSTP1-1 are in various stages of development, including one in Phase III for non-small cell lung cancer and ovarian cancer.
Curcumin
Nontraditional or alternative medicine is becoming an increasingly attractive approach for the treatment of various inflammatory disorders. Among these alternative approaches is the use of food derivatives, which have the advantage of being relatively nontoxic. A number of dietary compounds such as flavonoids and curcumin block activation of NFkB (Yamamoto et al., J. Clin. Invest., 107, 135 (2001); Bharti et al., Blood 101, 1053 (2003)). Curcumin is a non-nutritive, non-toxic polyphenol natural product found in turmeric, a spice that has been used for centuries in India and elsewhere as an herbal medicinal treatment of wounds, jaundice, and rheumatoid arthritis (Ammon et al., Planta Med., 57, 1 (1991)). Curcumin is the major constituent of turmeric powder extracted from the rhizomes of the plant Curcuma longa L found in south and southeast tropical Asia (Govindaraja, V. S., Crit. Rev. Food Sci. Nutri. 12:199 (1980)). In the countries of its origin, turmeric has also been used for centuries as a traditional medicine to treat inflammatory disorders. Scientists have subsequently demonstrated the anti-inflammatory properties of curcumin (Ammon et al., Planta Med. 57:1 (1991). Curcumin also exhibits potent anti-oxidant activity, which depends upon the presence of phenolic groups in the aryl rings (Baldwin, A. S. J. Clin. Invest. 107:241 (2001)). In traditional Indian medicine, curcumin has been used to treat a host of ailments through topical, oral and inhalation administration, and has recently been found safe in six human trials at oral loads up to 8 grams/day for 6 months. Chainani-Wu (2003) J Altern Complement Med 9, 161-168. Most of the clinical trials of curcumin pertain to its anti-tumor activity in colon, skin, stomach, duodenal, soft palate and breast cancers. However, the mechanism of action for curcumin is not well understood.
Curcumin derivatives have been shown to provide antitumor activity. For example, the antitumor activity of curcumin derivatives is described in U.S. patent application Ser. No. 11/057,636, entitled “Method and Compounds for Cancer Treatment Utilizing NFkB as a Direct or Ultimate Target for Small Molecule Inhibitors,” filed Feb. 14, 2005, by Vander Jagt et al. and incorporated herein by reference, and U.S. patent application Ser. No. 11/373,444, entitled “Cancer Treatment Using Curcumin Derivatives,” filed Mar. 10, 2006, also by Vander Jagt et al. and incorporated herein by reference.
Curcumin is a natural chemoprotective agent that elevates the activities of Phase 2 detoxification enzymes, while inhibiting procarcinogen activating Phase 1 enzymes. It decreases expression of several proto-oncogenes including c-jun, c-fos, and c-myc, and of particular interest, it suppresses the activation of NFκB. Related to this, curcumin has also been shown to induce apoptosis in several tumor cell lines. In addition to the down-regulation of uPA by dominant negative inhibitors of NFκB, numerous other factors, including VEGF, IL-8, and MMP-9 that contribute to angiogenesis, invasion, and metastasis are down-regulated by dominant negative inhibitors of NFκB. Likewise, curcumin inhibits angiogenesis in vivo. Curcumin can be viewed as a lead compound that inhibits metastasis and promotes apoptosis. Other antiangiogenic properties of curcumin are also known. Shim et al. have shown that curcumin causes the irreversible inhibition of CD13/aminopeptidase N, a membrane-bound, zinc-dependent metalloproteinase that plays a key role in tumor invasion and angiogenesis. Shim et al., “Irreversible inhibition of CD13/aminopeptidase N by the antigenic agent curcumin”, Chem. Biol. 10(8): 695-704 (August 2003).
Curcumin is known to inhibit the formation of Jun-Fos heterodimers in TPA induced cells and curcumin analogs are known to be up to 90 times more potent than curcumin (Hahm et al., Cancer Lett. 184, 89-96 (2002). It is also known that besides curcumin (turmeric), several natural products including resveratrol (peanuts and grape skins) (Manna et al., J. Immunol. 164, 6509-6519 (2000)), silymarin (artichoke) (Manna et al., J. Immunol. 163(12), 6800-6809 (1999)), oleandrin (Manna et al., Cancer Res. 60, 3838-3847 (2000)) and several compounds isolated from both green and black tea leaves (Chung et al., Cancer Res. 59, 4610-4617 (1999)) inhibit the AP-1 activation cascade. It is possible that curcumin analogs exhibit their activities on JNK since it is known that both silymarin (Manna et al., J. Immunol., 163(12), 6800-6809 (1999)) and oleandrin (Manna et al., Cancer Res. 60, 3838-3847 (2000)) inhibit JNK activity.
In addition, curcumin exhibits anti-inflammatory activity and is a potent anti-oxidant and free radical scavenger. Leu et al., (2002) Curr Med Chem Anti-Canc Agents 2, 357-370. In APP-overexpressing transgenic mice, curcumin reduced levels of oxidized proteins and inflammatory cytokine IL1 (Lim et al., (2001) J Neurosci 2, 8370-8377), thus offering a potential therapy against microglial activation in patients with Alzheimer's disease. Curcumin has additional activities of interest: it limits the progression of renal lesions in the STZ-diabetic rat model (Suresh Babu et al., (1998) Mol Cell Biochem 181, 87-96), and ameliorates oxidative stress-induced renal injury in mice (Okada et al., (2001) J Nutr 131, 2090-2095). Consequently, there has been extensive interest in the anti-oxidant properties of curcumin and the possibility that many of its biological activities are derived from its anti-oxidant properties. Balasubramanyam et al., (2003) J Biosci 28, 715-721.
Curcumin also inhibits the activation of NFκB (Bharti et al., (2003) Blood 101, 1053-1062), which may explain its anti-inflammatory properties. Curcumin was shown to attenuate the plasma inflammatory cytokine surge and cardiomyocyte apoptosis following cardiac ischemia/reperfusion in experimental animals by inhibiting activation of NFκB. Yeh et al., (2005) J Surg Res 125, 109-110. Curcumin suppressed NOS induction in LPS-stimulated macrophages by inhibiting the activation of NFκB. Pan et al., (2000) Biochem Pharmacol 60, 1655-1676. Likewise, curcumin inhibited mitogen stimulation of lymphocyte proliferation by inhibiting activation of NFκB. Ranjan et al., (2004) J Surg Res 121, 171-177. Of particular interest is the report that curcumin inhibits the activation of NFκB in BV2 microglia cells (Kang et al, (2004) J Pharmacol Sci 94, 325-328). The limited bioavailability of curcumin (Garcea et al., (2004) Br J Cancer 90, 1011-1015) suggests that clinical use of this natural product will be limited and points to the need to develop curcumin analogs with improved properties including improved bioavailability.
It was reported that curcumin inhibits TNF-α-induced NF-κB activation in human myelomonoblastic leukemia cells and phorbol ester-induced c-Jun/AP-1 activation in mouse fibroblast cells (Singh et al., J. Biol. Chem. 270:24995 (1995); Huang et al., Proc. Natl. Acad. Sci. USA 88:5292 (1991). The molecular mechanism for NF-κB inhibition by curcumin was unclear, but involved inhibition of I-κB degradation (Kumar et al., Biochem. Pharmacol. 55:775 (1998). More recent work has demonstrated that curcumin blocks intestinal endothelial cell gene expression by inhibiting the signal leading to IKK activation without directly interfering with NIK or IKK, and that blockade of IKK activation causes inhibition of I-κB phosphorylation/degradation and NF-κB activation (Jobin et al., J. Immunol. 163, 3474-83 (1999)).
The anti-inflammatory properties of curcumin and its ability to inhibit the immune response upon exposure to a variety of external stimuli may, at least in part, result from inhibition of the activation of NF-κB by these external signals, since many of the genes that are implicated in the immune/inflammatory response are up-regulated by NFκB. For example, curcumin inhibits the LPS-induced production of IL-1β and TNFα (Chan, M. M. Biochem. Pharmacol. 49, 1551 (1995)) and the IL-1β-induced expression of IL-2 (Chaudhary, L. R.; Avioli, L. V. J. Biol. Chem. 271, 16591 (1996)), as well as the TNFα-induced expression of ICAM-1, VCAM-1 and E-selectin (Gupta, B.; Ghosh, B. Int. J. Immunopharmacol. 21, 745 (1999)). NF-κB is implicated in these signaling pathways (Wang et al., Cytokine 29, 245 (2005); Krunkosky et al., Free Radical Biol. Med. 35, 1158 (2003)). However, curcumin has also been shown to be a direct inhibitor of enzymes that are important in the inflammatory response, including lipoxygenase and cyclo-oxygenase (Skrzypczak-Jankun et al., J. Int. J. Mol. Med. 6, 521 (2000)).
Further, curcumin has been shown to have possible application in the treatment of cystic fibrosis defects caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR), particularly for A508 mutations. Egan, et al., “Curcumin, A Major Constituent of Turmeric, Corrects Cystic Fibrosis Defects”, Science, 304: 600-602 (23 Apr. 2004).
The large consumption of curcumin by the Indian population may help explain their relatively low (4 times less) incidence of Alzheimer's disease compared to the U.S. population. Chandra et al., (2001) Neurology 57, 985-989. Although no systematic trials have been preformed using curcumin in India, recent studies have provided valuable insights on curcumin's role in Alzheimer's disease. Yang et al., (2005) J Biol Chem 280, 5892-5901; Ono et al., (2004) J Neurosci Res 75, 742-750. Curcumin was shown to inhibit the formation of Aβ oligomers and fibrils in vitro and reduce Aβ amyloid burden in vivo. Specifically, Ono et al. have indicated that curcumin inhibits the accumulation of amyloid β-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ and destabilizes preformed fAβ. Ono et al., “Curcumin Has Potent Anti-Amyloidogenic Effects for Alzheimer's β-Amyloid Fibrils In Vitro”, J. Neuroscience Res., 75: 742-750 (2004). Importantly, curcumin administered by intravenous (i.v.) injection lowered Aβ deposition in aged APP(Swedish)-transgenic mice (Tg2576), clearly demonstrating its ability to cross the blood-brain barrier in sufficient quantities to reduce amyloid burden. Curcumin is structurally similar to other inhibitors of Aβ aggregation such as Congo Red and Chrysamine G.
Thus, NFκB and its upstream regulators, as well as AP-1 and GSTP1-1, present inviting targets for development of anti-inflammatory drugs, and curcumin represents a promising lead compound. Analogues of curcumin that function as small molecules inhibitors of NFκB, AP-1 and GSTP1-1 activation are highly desirable for the treatment of diseases with inflammatory symptoms or components such as Alzheimer's disease, diabetes, cystic fibrosis and cancer, and also as assistive or adjuvant agents in the chemotherapeutic treatment of cancer.